摘要Alzheimer's disease (AD) is characterized by complex etiology, long-lasting pathogenesis, and cell-type-specific alterations. Currently, there is no cure for AD, emphasizing the urgent need for a comprehensive understanding of cell-specific pathology. Astrocytes, principal homeostatic cells of the central nervous system, are key players in the pathogenesis of neurodegenerative diseases, including AD. Cellular models greatly facilitate the investigation of cell-specific pathological alterations and the dissection of molecular mechanisms and pathways. Tumor-derived and immortalized astrocytic cell lines, alongside the emerging technology of adult induced pluripotent stem cells, are widely used to study cellular dysfunction in AD. Surprisingly, no stable cell lines were available from genetic mouse AD models. Recently, we established immortalized hippocampal astroglial cell lines from amyloid-β precursor protein/presenilin-1/Tau triple-transgenic (3xTg)-AD mice (denominated as wild type (WT)- and 3Tg-iAstro cells) using retrovirus-mediated transduction of simian virus 40 large T-antigen and propagation without clonal selection, thereby maintaining natural heterogeneity of primary cultures. Several groups have successfully used 3Tg-iAstro cells for single-cell and omics approaches to study astrocytic AD-related alterations of calcium signaling, mitochondrial dysfunctions, disproteostasis, altered homeostatic and signaling support to neurons, and blood-brain barrier models. Here we provide a comparative overview of the most used models to study astrocytes in vitro, such as primary culture, tumor-derived cell lines, immortalized astroglial cell lines, and induced pluripotent stem cell-derived astrocytes. We conclude that immortalized WT- and 3Tg-iAstro cells provide a non-competitive but complementary, low-cost, easy-to-handle, and versatile cellular model for dissection of astrocyte-specific AD-related alterations and preclinical drug discovery. Key Words: Alzheimer's disease; astrocytes immortalization; astroglial Alzheimers's disease model; blood-brain barrier; calcium signaling; central nervous system homeostasis; disproteostasis;endoplasmic reticulum-mitochondria contacts; induced pluripotent stem cell-derived astrocytes;protein synthesis Introduction The progressive increase of Alzheimer's disease (AD) incidence in the older adult population underlines the need for new disease-modifying therapeutic strategies to contrast neuronal degeneration (Merlo et al., 2021; Cummings, 2022). Alterations of Ca2+ homeostasis, mitochondrial dysfunction, oxidative stress, dysproteostasis, and cell-cell communication are characteristic of cellular dysfunction in AD. The neuronal contribution has been widely documented, and numerous neuronal in vitro models have been described, such as neuronal cell lines, primary neuronal cultures, and recent induced pluripotent stem cell (iPSC)-derived models (Chang et al., 2020). However, in the last 20 years, the role of astroglial cells in AD pathology, with the involvement of the mechanisms mentioned above, has received growing attention (Verkhratsky et al., 2012, 2021; Lim et al., 2016b). Astrocytes are the main homeostatic cells in the central nervous system (CNS), whose activity plays a crucial role in neuronal functions. Astrocytes provide metabolic support, ions, and neurotransmitter modulation and control extracellular environment and synaptic and blood-brain barrier (BBB) functions (Lim et al., 2016a; Adamsky and Goshen, 2018). Reports of recent decades brought a better understanding of their pathological contribution to neurodegenerative diseases. Indeed, astrocytes undergo complex yet brain region-specific and disease-stage-dependent alterations. Functional imaging studies on humans and works on animal models suggest that in early, preclinical, and prodromal disease stages, astrocytes become hypotrophic and asthenic (Verkhratsky et al., 2012, 2021). The asthenia is accompanied by a decrease in glucose utilization, reduction of glial fibrillary acidic protein (GFAP) immunoreactivity, and decrease in cell volume, suggesting reduced homeostatic support to neurons and other cells in the CNS. Another astrocytic disease state is represented by astrogliosis, characterized by hypertrophic changes, such as increased GFAP expression, microglial activation, and neuroinflammation. Astrogliosis is characteristic of advanced stages of many neurological and neurodegenerative diseases, including AD (Alberdi et al., 2013; Lim et al., 2016b, 2021; Verkhratsky et al., 2021; Sims et al., 2022). It involves transcriptional, proteomic, and functional remodeling of astrocytes with still unclear meaning for disease outcomes (Boulay et al., 2017; Garcia-Esparcia et al., 2017; Sakers et al., 2017; Escartin et al., 2021; Sims et al., 2022). These findings emphasize an urgent need for comprehensive research studies on astrocytes to dissect these cells' contribution to AD progression and discover new therapeutic targets (Cummings, 2022). Detailed investigation of astrocytic cellular pathophysiology in AD requires the development of cellular models which combine several apparently incompatible features, such as i) maintenance of cell-type specificity; ii) faithful reproduction of AD-specific cellular pathophysiology; iii) stability and scalability of cultures to be suitable for a range of assays from single cell to high throughput analyses, and iv) suitability for the inter-laboratory logistics, cryopreservation, and transportation. Several in vitro astroglial models have been used to study AD cellular dysfunction, such as primary astroglial cultures, tumor-derived astroglial cell lines, immortalized astrocytes, and iPSC-derived astrocytes. Surprisingly, no stable cell lines from mouse AD models were available. To fulfill this gap, we have recently generated immortalized hippocampal astroglial cell lines from amyloid-β (Aβ) precursor protein/presenilin-1/Tau triple-transgenic (3xTg)-AD mice (denominated as wild type (WT)- and 3Tg-iAstro cells) (Rocchio et al., 2019). iAstro lines were generated by retrovirus-mediated transduction of simian virus 40 (SV40) large T-antigen and propagated without clonal selection, thereby maintaining the natural heterogeneity of primary cultures. Here we discuss the main 1Department of Pharmaceutical Sciences, Università del Piemonte Orientale "Amedeo Avogadro", Novara, Italy; 2Department of Neuroscience, Mario Negri Institute forPharmacological Research IRCCS, Milano, Italy *Correspondence to: Laura Tapella, PhD, laura.tapella@uniupo.it; Dmitry Lim, PhD, dmitry.lim@uniupo.it. https://orcid.org/0000-0002-8159-1628 (Laura Tapella); https://orcid.org/0000-0002-4316-2654 (Dmitry Lim); https://orcid.org/0000-0003-2818-9039 (Massimiliano De Paola); https://orcid.org/0000-0002-6317-3182 (Giula Dematteis); https://orcid.org/0000-0003-1923-7430 (Armando A Genazzani) Funding: This work was supported by fellowship to a grant from CRT Foundation, No. 1393-2017 (to LT); grants from the Fondazione Cariplo, Nos. 2013-0795 (to AAG), 2014-1094 (to DL); and grants from The Università del Piemonte Orientale, Nos. FAR-2016 (to DL), FAR-2019 (to DL). How to cite this article: Tapella L, Dematteis G, Genazzani AA, De Paola M, Lim D (2023) Immortalized hippocampal astrocytes from 3xTg-AD mice, a new model to study disease- related astrocytic dysfunction: a comparative review. Neural Regen Res 18(8):1672-1678. From the Contents Introduction 1672 Astroglial In Vitro Models, Advantages and Disadvantages: Primary Cultures, Cell Lines, Immortalized Cell Lines and Induced Pluripotent Stem Cells 1673 Conclusions and Perspectives 1676 https://doi.org/10.4103/1673-5374.363192 Date of submission: September 11, 2022 Date of decision: October 11, 2022 Date of acceptance: November 8, 2022 Date of web publication: December 21, 2022 Laura Tapella1, *, Giulia Dematteis1, Armando A Genazzani1, Massimiliano De Paola2, Dmitry Lim1, *